p-xylene

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-06 to 2018-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented guideline study, with GLP and no significant deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

Guideline for Testing of Chemicals -“Earthworm, Reproduction Test (Eisenia fetida/Eisenia andrei)”, acc. to OECD Guideline 222 (July 2016)

Deviations:

yes

Remarks:

Timing of feeding changed and slight deviation in soil moisture.

Principles of method if other than guideline:

Deviations from guidelines: Food was provided on day 0 (start of exposure) instead of day 1 due to good experience with this procedure proved by more than 50 studies successfully completed with this method. The soil moisture slightly deviated by more than 10 % but less than 20 % from the initial value at the control and the test item concentrations 10 to 189 mg a.s./kg soil dry weight. These deviations are considered to have no impact on quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: Xylenes (Mixed Isomers)
Supplier: Sigma Aldrich
Batch number: SHBJ3208
CAS RN (a.s.): 1330-20-7
Molecular formula: C8H10
Molecular weight: 106.17 g/mol
Content (certified): 99.2 % mixed xylenes (83.2 % xylene isomers and 16.0 % ethylbenzene)
Density: Not specified
Appearance: Colourless liquid
Water solubility: Not specified
Expiry date: 2022-10-16

Analytical monitoring:

yes

Details on sampling:

Measurements and observation
Analytical evaluation of the test item was carried out via GC-MS/MS for all test item concentrations and the control at beginning of the experiment (day 0). Additionally, the test item concentrations of 32, 105 and 340 mg a.s./kg soil dry weight and the control were verified via GC-MS/MS at day -1, day 7 and day 28.

At the start and end of the exposure, the pH-value and moisture content of the test medium were determined in the control and each treatment from pooled samples of all biological replicates. The water content of each test container was checked weekly by weighing the test containers. The weight loss was replenished with the appropriate amount of demineralised water.

Details on preparation and application of test substrate:

Application rates: Nominal concentrations of 10, 18, 32, 58, 105, 189, 340, 612 mg/kg soil dry weight corresponding to the initial measured test item concentrations of 3.32, 7.42, 7.49, 26.3, 42.6, 93.1, 152, 334 mg/kg soil dry weight, respectively.
Control: Artificial soil moistened with demineralised water, without test or reference item will be used as control medium.

Test medium:
Artificial soil consisted of the following components:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.18 % calcium carbonate (CaCO3) to achieve a pH of 6.0  0.5

The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted. Five days before test start the artificial soil (31000 g) was pre-moistened with 2976 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

Application: Prior to application, the artificial soil was stored at 6 +/- 2°C for about 16 hours. On day -1, the respective test item amount for each concentration was pipetted into a graduated flask and mixed with demineralised water to receive an amount of 100 mL. These 100 ml were evenly distributed into 5 holes that were poked into the respective pre-moistened batch of soil. After addition of the test item, the holes were covered up with the surrounding soil and additional demineralised water was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax, each. Subsequently, the test medium was mixed thoroughly for 3 minutes with a handheld mixer and a stainless steel spoon to ensure a homogenous distribution. The vessels containing the different batches of spiked soil were closed and stored at 20 +/-2 °C for 24 hours. On day 0, the test medium was mixed once with a spoon and the amount of soil corresponding to approximately 600 g soil dry weight was filled into the test vessels, each.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test system: Eisenia fetida (Annelida, Lumbricidae)
Reason for the selection: Eisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline. This test system meets the requirements of the Regulation (EC) No. 1107/2009
Source: Breeding stock culture maintained at the test facility
Breeding: E. fetida are bred at the test facility in covered plastic vessels containing potting compost.
Synchronisation of the earthworm population: Earthworms of homogeneous age and body weight were used in the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.
Feeding: During breeding, the earthworms are fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.
Number and age of the earthworms per replicate: 10 adult earthworms (with clitellum) aged 2-12 months were used per replicate. The difference of age did not deviate by more than 1 month.
Body weight (measured): 0.34 to 0.56 g
Acclimation: Earthworms were acclimatised to the artificial soil to be used for the test two days prior to the start of exposure.
Homogeneity of groups: The worms were washed with demineralised water and dried gently on a paper towel. The homogeneity of the population was checked by weighing the individual earthworms to confirm they are within the required body weight range. After confirmation of homogeneity, groups of 10 earthworms (chosen by a randomised procedure) were weighed and introduced into each test vessel.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

17-22 °C

pH:

5.35 - 6.41

Moisture:

20.5 - 24.0 % of dry weight

Details on test conditions:

Application rates: Nominal concentrations of 10, 18, 32, 58, 105, 189, 340, 612 mg/kg soil dry weight corresponding to the initial measured test item concentrations of 3.32, 7.42, 7.49, 26.3, 42.6, 93.1, 152, 334 mg/kg soil dry weight, respectively.
Control: Artificial soil moistened with demineralised water, without test or reference item will be used as control medium.
Test duration: 8 weeks
Number of replicates: 8 replicates per control, 4 replicates per test item concentration and 1 additional replicate for analytical determination of the concentration levels 32, 105 and 340 mg/kg soil dry weight and the control (each replicate containing 10 earthworms).
Test container: Glass dishes (volume 1.5 L, wide mouth, mason jar) were used. During the test they were covered with opaque aluminium foil to prevent the test medium from drying as well as to minimize the volatility of the test substance. The foil was perforated on day 7 with 5 pin-sized holes per replicate to allow air exchange.
Temperature: 20 +/- 2 °C
Photoperiod: 16 h (light) : 8 h (dark)
Light intensity: 400 - 800 lx
Moistening: Throughout the test the replicates were weighed weekly. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test

Measurements and observation
At the start and end of the exposure, the pH-value and moisture content of the test medium were determined in the control and each treatment from pooled samples of all biological replicates. The water content of each test container was checked weekly by weighing the test containers. The weight loss was replenished with the appropriate amount of demineralised water. The body weight of the adult earthworms was determined individually on day 0 and day 28 for each replicate. On day 28, the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each replicate. After a further 28 days, the number of offspring (juveniles) hatched from the cocoons and the number of unhatched cocoons were counted for each replicate.

Observation parameters
A) no obvious pathological symptoms
B) no reaction to physical stimuli
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms

Equipment
pH-Meter, pH 3110, WTW
Hygrothermograph, LUFFT, LAMBRECHT
Drying cabinet, BINDER GmbH
Analytical balances, SARTORIUS
Balances, KERN
Waterbath, MEMMERT
Automatic Timer
Mixer, Dynamic model FT97
Mixer, SEVERIN
Luxmeter, ATP MESSTECHNIK
Positive displacement pipettes, GILSON

Statistics
Adult Mortality: Significant differences in adult mortality were determined in comparison with the control group by Step-down Cochran-Armitage Test Procedure. When running the test, a Qualitative Trend analysis by Contrasts (Monotonicity of Concentration/Response) and Tarone’s Test Procedure were done first. The LC10/20/50-values for mortality were calculated.
Biomass, Reproduction: Significant differences in biomass and reproduction were determined in comparison with the control group by Williams Multiple Sequential t-test Procedure. When running the tests, a Shapiro-Wilk’s Test on Normal Distribution and a Levene’s Test on Variance Homogeneity (with Residuals) were done first. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10/20/50-values for reproduction were calculated by probit analysis.

Software: The data presented in the tables of the report were computer-generated and have been rounded for presentation. Thus, manual re-calculation of the data based on the results presented in this report may result in minor deviations from these figures. Calculations were carried out using software:
- Excel, MICROSOFT
- ToxRat Professional, TOXRAT SOLUTIONS GMBH
- Sigma Plot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Nominal and measured concentrations:

Nominal concentrations = 0, 10, 18, 32, 58, 105, 189, 340, 612 mg/kg soil dry weight.
Initial measured test item concentrations = 3.32, 7.42, 7.49, 26.3, 42.6, 93.1, 152, 334 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

152 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

> 93.1 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: biomass

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

93.1 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

93.1 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 93.1 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: biomass

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

42.6 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

LC10

Effect conc.:

88.8 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 wk

Dose descriptor:

other: LC20

Effect conc.:

98.3 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 wk

Dose descriptor:

LC50

Effect conc.:

117 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

70.8 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

other: EC20

Effect conc.:

78.4 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

93.2 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Adult Mortality
No evident earthworm mortalities (< 10 %) were observed in the control or nominal concentrations 10 to 189 mg a.s./kg soil dry weight. However, at the nominal concentrations 340 and 612 mg a.s./kg soil dry weight 100 % mortality was observed. The initial measured LC50 – value for mortality was determined to be 117 mg a.s./kg soil dry weight.

Pathological Symptoms and Changes in Behaviour
No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or nominal concentrations 10 to 189 mg a.s. sum of xylenes isomers/kg soil dry weight. However, at the nominal concentrations 340 and 612 mg a.s. sum of xylenes isomers/kg soil dry weight 100 % mortality were observed. A statistically significant mortality of 5 % observed at the nominal concentration of 189 mg a.s mixed xylenes isomers/kg soil dry weight was below the biological threshold of 20 %.

Live Weight
At beginning of the test, the earthworms had individual weights in the recommended range of 0.34 - 0.56 g. There were no statistically significant differences in earthworm body weights in the nominal concentrations 10 to 189 mg a.s. sum of xylenes isomers/kg soil dry weight compared to the control. Due to 100 % mortality at the nominal concentrations 340 and 612 mg a.s. sum of xylenes isomers/kg soil dry weight no body weights were measured.

Reproduction Rate
The reproduction rate (average number of juveniles produced per replicate) was 152 in the control and ranged from 0 to 174 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the nominal treatment rates 10 to 105 mg a.s. sum of xylenes isomers/kg soil dry weight compared to the control. However, at the nominal concentrations 189, 340 and 612 mg a.s. sum of xylenes isomers/kg soil dry weight the earthworm reproduction was statistically significantly reduced. In the nominal concentration 340 and 612 mg a.s. sum of xylenes isomers/kg soil dry weight no juveniles were found at all. The number of unhatched cocoons ranged from 0 to 3 per replicate in the control and from 0 to 4 in the test item concentrations, respectively. The initial measured EC50-value for reproduction was determined to be 93.2 mg a.s. sum of xylenes isomers/kg soil dry weight. The coefficient of variation calculated for the reproduction of the control was 17.0 % and thus lower than the 30 %, required for a valid test.

Measured Exposure Concentrations during the Definitive Test
Analytical evaluation of the test item Xylenes (Mixed Isomers) was carried out via GC-MS/MS for all test item concentrations and the control at test start. Additionally, the concentrations of 38.2, 125 and 405 mg a.s. mixed xylenes / kg soil dry weight and the control were analyzed via GC-MS/MS at day -1, at day 7 and at day 28. The recovery rates at day -1 were between 44 and 62% and at day 0 between 20 and 46% of the nominal concentrations. After 7 days, recovery rates were between < LOQ and 12% and after 28 days, recovery rates were all < LOQ. All control samples were < LOQ.

Validity Criteria
This study on the effects of Xylenes (Mixed Isomers) on the reproduction of earthworms of the species Eisenia fetida was performed according to OECD Guidelines 222 (July 2016). All validity criteria were fulfilled:
- The adult mortality in the control group did not exceed 10 % after the first four weeks of the definitive test.
- The average number of juveniles in the control group was higher than 30 per replicate.
- The coefficient of variation calculated for the reproduction of the control was lower than 30 % at the end of the test.

Results with reference substance (positive control):

Reference item: CARBENDAZIM
Time schedule: The reference test with carbendazim is conducted once within twelve months. The most recent test was carried out from 2017-02-09 to 2017-04-13 (Study no. RBR71702).
Kind of application: Application into the artificial soil by mixing
Test concentration: 0, 0.5, 1, 2, 4 mg/kg soil dry weight
Purpose: To determine the sensitivity of the stock culture to the reference item. According to the guideline, earthworms are expected to show significant effects between 1 and 5 mg/kg soil dry weight.

Reported statistics and error estimates:

Adult Mortality: Significant differences in adult mortality were determined in comparison with the control group by Step-down Cochran-Armitage Test Procedure. When running the test, a Qualitative Trend analysis by Contrasts (Monotonicity of Concentration/Response) and Tarone’s Test Procedure were done first. The LC10/20/50-values for mortality were calculated.
Biomass, Reproduction: Significant differences in biomass and reproduction were determined in comparison with the control group by Williams Multiple Sequential t-test Procedure. When running the tests, a Shapiro-Wilk’s Test on Normal Distribution and a Levene’s Test on Variance Homogeneity (with Residuals) were done first. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10/20/50-values for reproduction were calculated by probit analysis.

Table 4. Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Nominal application rate of sum of xylenes isomers	Replicate
[mg/kg soil dry weight]	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
10	0	0	0	0	-	-	-	-	0
18	0	10	0	0	-	-	-	-	2.5
32	0	0	0	0	-	-	-	-	0
58	0	0	0	0	-	-	-	-	0
105	0	0	0	0	-	-	-	-	0
189	10	0	10	0	-	-	-	-	5^#
340	100	100	100	100	-	-	-	-	100*
612	100	100	100	100	-	-	-	-	100*

*) statistically significant compared to control (Step-down Cochran-Armitage Test Procedure).

   Although not 28 d observation, it was apparent that mortality had occurred by day 14.

#) statistically significant but below the biological threshold of 20 %

 

Table 5. Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Nominal application rate mixed xylene isomers	Replicate
[mg/kg soil dry weight]	Observation parameter	1	2	3	4	5	6	7	8
Control	A	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
10	A	10/10	10/10	10/10	10/10	–	–	–	–
18	A H	10/10 -	9/10 1/10	10/10 -	10/10 -	–	–	–	–
32	A	10/10	10/10	10/10	10/10	–	–	–	–
58	A	10/10	10/10	10/10	10/10	–	–	–	–
105	A	10/10	10/10	10/10	10/10	–	–	–	–
189	A H	9/10 1/10	10/10 -	9/10 1/10	10/10 -	–	–	–	–
340	H	10/10	10/10	10/10	10/10	–	–	–	–
612	H	10/10	10/10	10/10	10/10	–	–	–	–

A = no obvious pathological symptoms      H = Earthworm dead

Table 6. Body weight changes of the adult earthworms.

Nominal application rate mixed xylene isomers [mg/kgsoil dry weight]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.44	0.54	0.10	22.7	0.11 ± 0.05	23.8 ± 10.7	–
	2	0.44	0.52	0.08	18.2
	3	0.43	0.62	0.19	44.2
	4	0.45	0.54	0.09	20.0
	5	0.44	0.56	0.12	27.3
	6	0.45	0.55	0.10	22.2
	7	0.45	0.48	0.03	6.7
	8	0.45	0.58	0.13	28.9
10	1	0.44	0.59	0.15	34.1	0.12 ± 0.04	26.5 ± 8.35	No
	2	0.45	0.52	0.07	15.6
	3	0.44	0.58	0.14	31.8
	4	0.45	0.56	0.11	24.4
18	1	0.44	0.58	0.14	31.8	0.12 ± 0.02	26.6 ± 5.88	No
	2	0.45	0.59	0.14	31.1
	3	0.46	0.55	0.09	19.6
	4	0.46	0.57	0.11	23.9
32	1	0.44	0.55	0.11	25.0	0.11 ± 0.03	24.4 ± 7.15	No
	2	0.44	0.53	0.09	20.5
	3	0.44	0.59	0.15	34.1
	4	0.45	0.53	0.08	17.8
58	1	0.43	0.51	0.08	18.6	0.10 ± 0.03	23.2 ± 5.84	No
	2	0.45	0.53	0.08	17.8
	3	0.44	0.57	0.13	29.5
	4	0.45	0.57	0.12	26.7
105	1	0.45	0.53	0.08	17.8	0.09 ± 0.02	20.2 ± 5.48	No
	2	0.45	0.56	0.11	24.4
	3	0.44	0.50	0.06	13.6
	4	0.44	0.55	0.11	25.0
189	1	0.44	0.64	0.20	45.5	0.16 ± 0.03	36.4 ± 7.65	No
	2	0.44	0.59	0.15	34.1
	3	0.44	0.61	0.17	38.6
	4	0.44	0.56	0.12	27.3
340	1	0.43	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	2	0.43	n.d.	n.d.	n.d.
	3	0.42	n.d.	n.d.	n.d.
	4	0.42	n.d.	n.d.	n.d.
612	1	0.43	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	2	0.42	n.d.	n.d.	n.d.
	3	0.42	n.d.	n.d.	n.d.
	4	0.43	n.d.	n.d.	n.d.

SD = Standard deviation          n.d. = not determined                           

Sig. = Statistical Significance (Williams Multiple Sequential t-test Procedure,α= 0.05) compared to control

Table 7. Reproduction Rate (Number of Juveniles after 8 Weeks)

Nominal application rate mixed xylene isomers [mg/kg soil dry weight]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] of control	Sig.
Control	1	154	152 ± 25.8	17.0	-	-
	2	143
	3	102
	4	153
	5	157
	6	154
	7	197
	8	156
10	1	119	148 ± 23.7	16.0	97.4	No
	2	166
	3	137
	4	168
18	1	193	174 ± 13.6	7.81	114	No
	2	162
	3	167
	4	174
32	1	131	150 ± 19.9	13.3	98.7	No
	2	137
	3	156
	4	175
58	1	158	163 ± 35.4	21.7	107	No
	2	153
	3	128
	4	212
105	1	178	163 ± 16.0	9.79	107	No
	2	148
	3	151
	4	176
189	1	48	76 ± 22.1	29.1	50.0	Yes
	2	83
	3	73
	4	101
340	1	0	0	0	0	Yes
	2	0
	3	0
	4	0
612	1	0	0	0	0	Yes
	2	0
	3	0
	4	0

Table 8. Number of unhatched Cocoons after 8 Weeks of Exposure

Nominal application rate mixed xylenes isomers	Replicate
[mg/kg soil dry weight]	1	2	3	4	5	6	7	8
Control	0	0	2	2	3	2	2	1
10	0	1	3	1	-	-	-	-
18	0	0	1	0	-	-	-	-
32	3	0	0	0	-	-	-	-
58	4	0	0	0	-	-	-	-
105	0	0	4	1	-	-	-	-
189	0	2	3	4	-	-	-	-
340	0	0	0	1	-	-	-	-
612	0	0	0	0	-	-	-	-

Table 9. Measrued concentrations and Percent of Nominal mixed xylene isomers.

Sampling date	Day -1 before exposure		Start of exposure		Day 7 after exposure		Day 28 after exposure
Nominal concentration mixed xylene isomers [mg/kg soil dry weight]	Meas. amount [mg/kg soil dry weight]	[%]	Meas. amount [mg/kg soil dry weight]	[%]	Meas. amount [mg/kg soil dry weight]	[%]	Meas. amount [mg/kg soil dry weight]	[%]
730	-	-	334	46	-	-	< LOQ	< LOQ
405	215	53	152	38	49.1	12	-	-
225	-	-	93.1	41	-	-	-	-
125	55.3	44	42.6	34	1.24	1.0	-	-
69.2	-	-	26.3	38	-	-	-	-
38.2	23.7	62	7.49	20	< LOQ	< LOQ	< LOQ	< LOQ
21.5	-	-	7.42	35	-	-	-	-
11.9	-	-	3.32	28	-	-	-	-
Control	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ	< LOQ

Meas.amount = Measured amount, enrichment and dilution factor taken into account

% = Percent of the nominal concentration of the a.s.mixed xylenes

LOQ = Limit of quantification of the analytical method (1.01 mg/kg soil dry weight)

- = not determined

Table 10. Reference Item -Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate carbendazim [mg/kg soil dry weight]	Rep.	Number of juveniles	Mean±SD	CV	% of control	Significance*
Control	1	170	152± 26.0	17.1	_	_
	2	141
	3	125
	4	123
	5	199
	6	167
	7	136
	8	156
0.5	1	182	153 ± 25.6	16.7	101	No
	2	167
	3	132
	4	131
1	1	128	145 ± 26.7	18.4	95.4	No
	2	154
	3	119
	4	178
2	1	10	6± 3.65	60.8	3.95	Yes
	2	8
	3	2
	4	4
4	1	0	0± 0.0	-	0.0	Yes
	2	0
	3	0
	4	0

Rep. = Replicate  a.s. = active substance  SD = Standard deviation      CV = Coefficient of variation

*) = Statistically significantly reduced compared tocontrol (Multiple Sequential-rejective Welsh-t-test After Bonferroni-Holm )

Validity criteria fulfilled:

yes

Conclusions:

During this study on the effects of mixed xylenes on earthworms, a NOEC (reproduction) of 42.6 mg/kg soil dry weight (based on initial measured concentrations, equivalent to a nominal 125 mg/kg soil dry weight) was determined based on significant reductions in the production of juveniles at the top three doses tested (initial measured concentrations of 93.1, 152 and 334 mg/kg soil dry weight). Following statistical analysis, an EC50 (reproduction) of 93.2 mg/kg soil dry weight (95 % confidence limits for range 87.4-99.3 mg/kg soil dry weight, based on the initial measured concentrations) was determined based on significant decreases in the presence of juveniles compared to controls.

Executive summary:

Effects of Xylenes(Mixed Xylenes) on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016).

In an 8 -week study, earthworm species Eisenia fetida were exposed to mixed xylene isomers in soil at nominal application rates of 0, 10, 18, 32, 58, 105, 189, 340 or 612 mg/kg soil dry weight (reported to be initial measured concentrations of 0, 3.32, 7.42, 7.49, 26.3, 42.6, 93.1, 152, and 334 mg/kg soil dry weight). Each application rate was mixed into the artificial soil containing 5 % peat one day prior to introducing earthworms to the soil. A control using untreated artificial soil was tested under the same conditions as the test item treatments. After approx. 24 hours of equilibration of the test item a total of 80 test organisms were introduced into 8 control replicates and 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.34 and 0.56 g at the beginning of the experiment. To reduce volatility, the test containers were covered with opaque aluminium foil which was perforated on day 7 with 5 pin-sized holes per replicate to allow air exchange.

Analytical evaluation of the test item mixed xylene isomers was carried out via GC-MS/MS for all test item concentrations and the control at beginning of the experiment (day 0). Additionally, the nominal test item application rates of 32, 105 and 340 mg/kg soil dry weight and the control were verified via GC-MS/MS at day -1, day 7 and day 28. The recovery rates at day -1 were between 44 and 62% and at day 0 between 20 and 46% of the nominal concentrations. After 7 days, recovery rates were between < LOQ and 12% and after 28 days, recovery rates were all < LOQ. All control samples were < LOQ. All effect levels were based on the measured initial values (day 0).

After 28 days of exposure, at the top two doses100 % mortality was observed. In soil at initial measured test item concentrations 0 to 93.1 mg/kg soil dry weight, no evident earthworm mortalities (< 10 %) or pathological symptoms or changes in the behaviour of adult earthworms were observed. Due to 100 % mortality at the initial measured test item concentrations top two doses no body weights were measured. There were no statistically significant differences in earthworm body weights in the initial measured test item concentrations 3.32 to 93.1 mg/kg soil dry weight compared to the control. After a further four weeks, the mean reproduction rate (average number of juveniles produced per replicate) was 152 in the control and ranged from 0 to 174 in the test item treatment rates. There were statistically significant decreases in the production of juveniles at the top three doses (reported to be initial measured test item concentrations 93.1, 152 and 334 mg/kg soil dry weight) and at the top two doses, no juveniles were found. There were no statistically significant differences in the production of juveniles in the remaining treatment rates (reported to be initial measured concentrations of 3.32 to 42.6 mg/kg soil dry weight) compared to the control.

A NOEC (reproduction) of 42.6 mg/kg soil dry weight (based on initial measured concentrations, equivalent to a nominal 125 mg/kg soil dry weight) was determined based on significant reductions in the production of juveniles at the top three doses tested (reported to be initial measured concentrations of 93.1, 152 and 334 mg/kg soil dry weight). Following statistical analysis, an EC50 (reproduction) of 93.2 mg/kg soil dry weight (95 % confidence limits for range 87.4-99.3 mg/kg soil dry weight, based on the initial measured concentrations) was determined based on significant decreases in the presence of juveniles compared to controls.

All validity criteria recommended by the test guidelines were fulfilled.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-06 to 2018-02-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented guideline study with GLP and no significant deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

OECD Guideline 222 (2016): Guideline for Testing of Chemicals-“Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei)”.

Deviations:

yes

Remarks:

Timing of feeding changed and slight deviation in soil moisture.

Principles of method if other than guideline:

Deviations from guidelines: Food was provided on day 0 (start of exposure) instead of day 1 due to good experience with this procedure proved by more than 50 studies successfully completed with this method. The soil moisture slightly deviated by more than 10 % but less than 20 % from the initial value at the control and the test item concentrations 10 to 189 mg a.s./kg soil dry weight. These deviations are considered to have no impact on quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: p-Xylene
Supplier: Merck
Batch number: S7428291
CAS RN (a.s.): 106-42-3
Molecular formula: C8H10
Molecular weight: 106.17 g/mol
Content (certified): 99.7 % (a/a)
Density (certified): 0.861 g/cm3 (d 20 °C/ 4 °C)
Appearance: Colourless liquid
Water solubility: 0.2 g/L at 25 °C
Expiry date: 2022-05-31

Analytical monitoring:

yes

Details on sampling:

Analytical evaluation of the test item p-Xylene was carried out via GC-MS/MS for all test item concentrations and the control at beginning of the experiment (day 0). Additionally, the test item concentrations 32, 105 and 340 mg a.s./kg soil dry weight and the control were verified via GC-MS/MS at day -1, day 7 and day 28.

At the start and the end of exposure, the pH-value and moisture content of the test medium were determined in the control and each treatment from pooled samples of all biological replicates. The water content of each test container was checked weekly by weighing the test containers. The weight loss was replenished with the appropriate amount of demineralised water.

Details on preparation and application of test substrate:

Application rates: 10, 18, 32, 58, 105, 189, 340 or 612 mg a.s./kg soil dry weight corresponding to the initial measured test item concentrations of 3.48, 7.04, 11.5, 22.2, 35.8, 71.1, 124 and 235 mg a.s./kg soil dry weight, respectively.
Control: Artificial soil moistened with demineralised water, without test or reference item was used as control medium.

Test medium
Artificial soil consisted of the following components:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.18 % calcium carbonate (CaCO3) to achieve a pH of 6.0 +/- 0.5

The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted. Five days before test start the artificial soil (31000 g) was pre-moistened with 2976 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

Application
Prior to application, the artificial soil was stored at 6 +/- 2°C for about 16 hours. On day -1, the respective test item amount for each concentration was pipetted into a graduated flask and mixed with demineralised water to receive an amount of 100 mL. These 100 ml were evenly distributed into 5 holes that were poked into the respective pre-moistened batch of soil. After addition of the test item, the holes were covered up with the surrounding soil and additional demineralised water was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax, each. Subsequently, the test medium was mixed thoroughly for 3 minutes with a handheld mixer and a stainless steel spoon to ensure a homogenous distribution. The vessels containing the different batches of spiked soil were closed and stored at 20 +/- 2 °C for 24 hours. On day 0, the test medium was mixed once with a spoon and the amount of soil corresponding to approximately 600 g soil DW was filled into the test vessels, each.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test system: Eisenia fetida (Annelida, Lumbricidae)
Reason for the selection: Eisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline. This test system meets the requirements of the Regulation (EC) No. 1107/2009
Source: Breeding stock culture maintained at the test facility
Breeding: E. fetida are bred at the test facility in covered plastic vessels containing potting compost.
Synchronisation of the earthworm population: Earthworms of homogeneous age and body weight were used in the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.
Feeding: During breeding, the earthworms are fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.
Number and age of the earthworms: 10 adult earthworms (with clitellum) aged 2-12 months were used per replicate. The difference of age did not deviate by more than 1 month (see “Synchronisation”).
Body weight (measured): 0.34 to 0.57 g
Acclimation: Earthworms were acclimatised to the artificial soil to be used for the test two days prior to the start of exposure.
Homogeneity of groups: The worms were washed with demineralised water and dried gently on a paper towel. The homogeneity of the population was checked by weighing the individual earthworms to confirm they are within the required body weight range. After confirmation of homogeneity, groups of 10 earthworms (chosen by a randomised procedure) were weighed and introduced into each test vessel.

Study type:

laboratory study

Substrate type:

artificial soil

Total exposure duration:

8 wk

Test temperature:

17-22 °C

pH:

5.51 - 6.37

Moisture:

20.4 - 23.8 % of dry weight

Details on test conditions:

Test duration: 8 weeks
Number of replicates: 8 replicates per control, 4 replicates per test item concentration and 1 additional replicate for analytical determination of the concentration levels 32, 105 and 340 mg a.s./kg soil dry weight and the control (each replicate containing 10 earthworms).
Test container: Glass dishes (volume 1.5 L, wide mouth, mason jar) were used. During the test they were covered with opaque aluminium foil to prevent the test medium from drying as well as to allow for the volatility of the test substance. The foil was perforated on day 7 with 5 pin-sized holes per replicate to allow air exchange.
Application: Prior to application, the artificial soil was stored at 6 +/- 2°C for about 16 hours. On day -1, the respective test item amount for each concentration was pipetted into a graduated flask and mixed with demineralised water to receive an amount of 100 mL. These 100 ml were evenly distributed into 5 holes that were poked into the respective pre-moistened batch of soil. After addition of the test item, the holes were covered up with the surrounding soil and additional demineralised water was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax, each. Subsequently, the test medium was mixed thoroughly for 3 minutes with a handheld mixer and a stainless steel spoon to ensure a homogenous distribution. The vessels containing the different batches of spiked soil were closed and stored at 20 +/- 2 °C for 24 hours. On day 0, the test medium was mixed once with a spoon and the amount of soil corresponding to approximately 600 g soil DW was filled into the test vessels, each.
Temperature: 20 +/- 2 °C
Photoperiod: 16 h (light) : 8 h (dark)
Light intensity: 400 - 800 lx
Feeding: The earthworms were fed 5 g of cattle manure (moistened with 5 mL of demineralised water) on a weekly basis during the first 4 weeks of the test. At day 28 the food was carefully mixed into the soil, on the other days the food was placed on the soil surface. Since all earthworms died before day 14 in the nominal test item concentrations 340 and 612 mg a.s./kg soil dry weight, no food and no additional demineralised water was added to these replicates from day 14 on. The cattle manure was delivered by LEHR- UND FORSCHUNGSGUT RUTHE, Schäferberg 1, 31157 Sarstedt, Germany. Manure was obtained from cattle that were not subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary products that could adversely affect the worms during the test. The unpasteurized manure was air-dried and finely ground before use.
Moistening: Throughout the test the replicates were weighed weekly. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test.

Measurements and observation
At the start and the end of exposure, the pH-value and moisture content of the test medium were determined in the control and each treatment from pooled samples of all biological replicates. The water content of each test container was checked weekly by weighing the test containers. The weight loss was replenished with the appropriate amount of demineralised water. The body weight of the adult earthworms was determined individually on day 0 and day 28 for each replicate. On day 28, the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each replicate. After a further 28 days, the number of offspring (juveniles) hatched from the cocoons and the number of unhatched cocoons were counted for each replicate.
 
Observation parameters
A) no obvious pathological symptoms
B) no reaction to physical stimuli
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms

Equipment
pH-Meter, pH 3110, WTW
Hygrothermograph, LUFFT, LAMBRECHT
Drying cabinet, BINDER GmbH
Analytical balances, SARTORIUS
Balances, KERN
Waterbath, MEMMERT
Automatic Timer
Mixer, Dynamic model FT97, THIEL GROßKÜCHENBEDARF
Mixer, SEVERIN
Luxmeter, TESTO
Positive displacement pipettes, GILSON

Reference test

Reference item: CARBENDAZIM
Time schedule: The reference test with carbendazim is conducted once within twelve months. The most recent test was carried out from 2017-02-09 to 2017-04-13 (Study no. RBR71702). The results are given in section 14.
Kind of application: Application into the artificial soil by mixing
Test concentration: 0.5 - 1 - 2 - 4 mg a.s./kg soil dry weight
Purpose: To determine the sensitivity of the stock culture to the reference item. According to the guideline, earthworms are expected to show significant effects between 1 and 5 mg a.s./kg soil dry weight.

Nominal and measured concentrations:

Analytical evaluation of the test item p-Xylene was carried out via GC-MS/MS for all test item concentrations and the control at test start. Additionally, the test item concentrations of 32, 105 and 340 mg a.s./kg soil dry weight and the control were analyzed via GC-MS/MS at day -1, at day 7 and at day 28. The recovery rates at day -1 were between 46 and 49 % and at day 0 between 34 and 39 % of the nominal concentrations. After 7 days, recovery rates were between < LOQ and 18 % and after 28 days all recovery rates were all < LOQ. All control samples were < LOQ.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

8 wk

Dose descriptor:

LOEC

Remarks:

reproduction, mortality

Effect conc.:

124 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

reproduction

Duration:

8 wk

Dose descriptor:

LOEC

Remarks:

biomass

Effect conc.:

> 71.1 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Remarks:

reproduction, mortality

Effect conc.:

71.1 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

reproduction

Duration:

8 wk

Dose descriptor:

NOEC

Remarks:

biomass

Effect conc.:

>= 71.1 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: biomass

Duration:

8 wk

Dose descriptor:

LC10

Remarks:

mortality

Effect conc.:

71.4 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% confidence interval (68.3-75.8 mg/kg dw)

Duration:

8 wk

Dose descriptor:

other: LC20

Remarks:

mortality

Effect conc.:

79 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% confidence interval, (75.5 – 83.6 mg/kg dw)

Duration:

8 wk

Dose descriptor:

LC50

Remarks:

mortality

Effect conc.:

93.9 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % Confidence Interval, (89.4 – 98.7 mg/kg dw)

Duration:

8 wk

Dose descriptor:

EC10

Remarks:

reproduction

Effect conc.:

67.5 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % Confidence Interval, (62.7 – 77.0 mg/kg dw)

Duration:

8 wk

Dose descriptor:

other: EC20

Remarks:

reproduction

Effect conc.:

74.7 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % Confidence Interval, (69.2 – 83.6 mg/kg bw)

Key result

Duration:

8 wk

Dose descriptor:

EC50

Remarks:

reproduction

Effect conc.:

88.8 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % Confidence Interval, (81.3 – 97.1 mg/kg bw)

Details on results:

Adult Mortality
No earthworm mortalities were observed in the control or nominal test item concentrations 10 to 189 mg a.s./kg soil dry weight. However, at the nominal test item concentration 340 and 612 mg a.s./kg soil dry weight 100 % mortality was observed. The initial measured LC50 – value for mortality was determined to be 93.9 mg a.s./kg soil dry weight.

Pathological Symptoms and Changes in Behaviour
No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or nominal test item concentrations 10 to 189 mg a.s./kg soil dry weight. However, at the nominal test item concentrations 340 and 612 mg a.s./kg soil dry weight 100 % mortality was observed.

Reproduction rate
The reproduction rate (average number of juveniles produced per replicate) was 177 in the control and ranged from 0 to 193 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the nominal treatment rates 10 to 189 mg a.s./kg soil dry weight compared to the control. However, at the nominal test item concentrations 340 and 612 mg a.s./kg soil dry weight the earthworm reproduction was statistically significantly reduced, with no juviniles found at these concentrations. The number of unhatched cocoons ranged from 0 to 6 per replicate in the control and from 0 to 12 in the test item concentrations, respectively. The initial measured EC50-value for reproduction was determined to be 88.8 mg a.s./kg soil dry weight. The coefficient of variation calculated for the reproduction of the control was 16.5 % and thus lower than the 30 %, required for a valid test.

Measured exposure concentrations during the definitive test
Analytical evaluation of the test item p-Xylene was carried out via GC-MS/MS for all test item concentrations and the control at test start. Additionally, the test item concentrations of 32, 105 and 340 mg a.s./kg soil dry weight and the control were analyzed via GC-MS/MS at day -1, at day 7 and at day 28. The recovery rates at day -1 were between 46 and 49 % and at day 0 between 34 and 39 % of the nominal concentrations. After 7 days, recovery rates were between < LOQ and 18 % and after 28 days all recovery rates were all < LOQ. All control samples were < LOQ.

Results with reference substance (positive control):

Reference item: ARBENDAZIM
Time schedule: The reference test with carbendazim is conducted once within twelve months. The most recent test was carried out from 2017-02-09 to 2017-04-13 (Study no. RBR71702). The results are given in section 14.
Kind of application: Application into the artificial soil by mixing
Test concentration: 0.5 - 1 - 2 - 4 mg a.s./kg soil dry weight
Purpose: To determine the sensitivity of the stock culture to the reference item. According to the guideline, earthworms are expected to show significant effects between 1 and 5 mg a.s./kg soil dry weight.

Reported statistics and error estimates:

Statistics and Calculations
Adult Mortality: Significant differences in adult mortality were determined in comparison with the control group by Step-down Cochran-Armitage Test Procedure. When running the test, a Qualitative Trend analysis by Contrasts (Monotonicity of Concentration/Response) and Tarone’s Test Procedure were done first. The LC10/20/50-values for mortality were calculated.

Biomass, Reproduction: Significant differences in biomass and reproduction were determined in comparison with the control group by Williams Multiple Sequential t-test Procedure (biomass) and Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm (reproduction), respectively. When running the tests, a Shapiro-Wilk’s Test on Normal Distribution and a Levene’s Test on Variance Homogeneity (with Residuals) were done first. Additionally, a Trend analysis was done for biomass values. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. The EC10/20/50-values for reproduction were calculated by probit analysis.

Table 3. Mortality of Adult Earthworms in [%] after 28 days of exposure.

Nominal application rate	Replicate
[mg a.s./kg SDW]	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
10	0	0	0	0	-	-	-	-	0
18	0	0	0	0	-	-	-	-	0
32	0	0	0	0	-	-	-	-	0
58	0	0	0	0	-	-	-	-	0
105	0	0	0	0	-	-	-	-	0
189	0	0	0	0	-	-	-	-	0
340	100	100	100	100	-	-	-	-	100*
612	100	100	100	100	-	-	-	-	100*

*) statistically significant compared to control (Step-down Cochran-Armitage Test Procedure). Although not 28 d observation it was apparent that mortality had occured by day 14.

Table 4. Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Nominal application rate	Replicate
[mg a.s./kg SDW]	Observation parameter	1	2	3	4	5	6	7	8
Control	A	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
10	A	10/10	10/10	10/10	10/10	–
18	A	10/10	10/10	10/10	10/10
32	A	10/10	10/10	10/10	10/10
58	A	10/10	10/10	10/10	10/10
105	A	10/10	10/10	10/10	10/10
189	A	10/10	10/10	10/10	10/10
340	H	10/10	10/10	10/10	10/10
612	H	10/10	10/10	10/10	10/10

A = no obvious pathological symptoms            H = Earthworm dead

Table 5. Body weight changes if the adult earthworms

Nominal application rate [mg a.s./kg SDW]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.46	0.53	0.07	15.2	0.08 ± 0.03	17.9 ± 6.09	–
	2	0.46	0.52	0.06	13.0
	3	0.45	0.57	0.12	26.7
	4	0.45	0.57	0.12	26.7
	5	0.46	0.54	0.08	17.4
	6	0.46	0.55	0.09	19.6
	7	0.46	0.51	0.05	10.9
	8	0.45	0.51	0.06	13.3
10	1	0.45	0.58	0.13	28.9	0.12 ± 0.02	26.4 ± 4.99	No
	2	0.45	0.59	0.14	31.1
	3	0.46	0.58	0.12	26.1
	4	0.46	0.55	0.09	19.6
18	1	0.46	0.56	0.10	21.7	0.10 ± 0.03	20.6 ± 5.49	No
	2	0.46	0.56	0.10	21.7
	3	0.46	0.58	0.12	26.1
	4	0.46	0.52	0.06	13.0
32	1	0.45	0.55	0.10	22.2	0.11 ± 0.01	23.2 ± 1.43	No
	2	0.45	0.56	0.11	24.4
	3	0.46	0.56	0.10	21.7
	4	0.45	0.56	0.11	24.4
58	1	0.46	0.53	0.07	15.2	0.10 ± 0.02	20.9 ± 5.21	No
	2	0.45	0.56	0.11	24.4
	3	0.46	0.58	0.12	26.1
	4	0.45	0.53	0.08	17.8
105	1	0.46	0.56	0.10	21.7	0.09 ± 0.01	20.3 ± 3.14	No
	2	0.45	0.56	0.11	24.4
	3	0.46	0.54	0.08	17.4
	4	0.45	0.53	0.08	17.8
189	1	0.46	0.58	0.12	26.1	0.10 ± 0.01	22.4 ± 3.14	No
	2	0.46	0.55	0.09	19.6
	3	0.46	0.57	0.11	23.9
	4	0.45	0.54	0.09	20.0
340	1	0.46	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	2	0.45	n.d.	n.d.	n.d.
	3	0.46	n.d.	n.d.	n.d.
	4	0.46	n.d.	n.d.	n.d.
612	1	0.45	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	2	0.46	n.d.	n.d.	n.d.
	3	0.45	n.d.	n.d.	n.d.
	4	0.46	n.d.	n.d.	n.d.

SD = Standard deviation                                           

Sig. = Statistical Significance (Williams Multiple Sequential t-test Procedure,α= 0.05) compared to control

Table 6. Reproduction rate (Number of Juveniles after 8 weeks)

Nominal application rate  [mga.s./kgSDW]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] of control	Sig.
Control	1	148	177 ± 29.1	16.5	-	-
	2	176
	3	198
	4	155
	5	223
	6	145
	7	206
	8	163
10	1	153	173 ± 13.3	7.69	97.7	No
	2	177
	3	177
	4	183
18	1	176	174 ± 15.3	8.82	98.3	No
	2	152
	3	181
	4	187
32	1	190	193 ± 16.2	8.40	109	No
	2	203
	3	207
	4	171
58	1	131	157 ± 22.1	14.1	88.7	No
	2	175
	3	147
	4	176
105	1	163	171 ± 10.7	6.26	96.6	No
	2	183
	3	177
	4	161
189	1	109	157 ± 32.6	20.7	88.7	No
	2	177
	3	178
	4	163
340	1	0	0	0	0	Yes
	2	0
	3	0
	4	0
612	1	0	0	0	0	Yes
	2	0
	3	0
	4	0

Table 7. Number of unhatched Cocoons after 8 weeks of exposure

Nominal application rate	Replicate
[mg a.s./kg SDW]	1	2	3	4	5	6	7	8
Control	0	1	2	0	1	6	5	0
10	1	9	7	1	-	-	-	-
18	1	2	1	0	-	-	-	-
32	0	2	1	1	-	-	-	-
58	5	12	7	7	-	-	-	-
105	3	4	0	0	-	-	-	-
189	4	1	1	2	-	-	-	-
340	0	0	0	0	-	-	-	-
612	0	0	1	0	-	-	-	-

Table 8. Measured concentration and percent of the nominal of p-xylene

Sampling date	Day -1 before exposure		Start of exposure		Day 7 after exposure		Day 28 after exposure
Nominal concentration [mg a.s./kg soil dry weight]	p-Xylene
	Meas. amount [mg a.s./kg soil dry weight]	[%]	Meas. amount [mg a.s./kg soil dry weight]	[%]	Meas. amount [mg a.s./kg soil dry weight]	[%]	Meas. amount [mg a.s./kg soil dry weight]	[%]
612	-		235	38	-
340	167	49	124	36	60.4	18	< LOQ
189	-		71.1	38	-
105	48.1	46	35.8	34	1.11	1.1	< LOQ
58	-		22.2	38	-
32	15.4	48	11.5	36	< LOQ		< LOQ
18	-		7.04	39	-
10	-		3.48	35	-
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas.amount= Measured amount, enrichment and dilution factor taken into account

% = Percent of the nominal concentration of the a.s.

LOQ =Limit of quantification of the analytical method (1.0 mg test item/kg SDW, corresponding to 0.997 mg a.s./kg SDW)

- = not determined

Table 9. pH Values, Moisture and WHCmax of the Test Media

Nominal application rate [mg a.s./kg SDW]	pH-value		Moisture [%] of dry weight		WHCmaxof artificial soil [g/100 g DW]
	day 0	day 57	day 0	day 57
Control	5.69	6.15	20.7	22.9*	39.1
10	5.63	6.21	20.4	23.5*
18	5.51	5.95	20.9	23.7*
32	5.67	6.14	20.9	23.5*
58	5.75	6.34	21.0	23.5*
105	5.58	6.22	21.1	23.8*
189	5.72	6.37	21.0	23.4*
340	5.57	6.09	20.5	21.7
612	5.82	6.18	20.7	22.1

WHC = Water Holding Capacity

*) Moisture slightly deviated by more than 10 % but less than 20 % from the initial value. This deviation is considered to have no impact on quality and integrity of the study.

Table 10. Environmental Conditions

	Room Temperature [°C]	Photoperiod [h]	Light intensity [lx]
nominal	20±2	16	400 - 800
actual	17 – 22	16	594 ± 106

Reference test.

Table 11. Reference Item Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg a.s./kg soil dry weight]	Rep.	Number of juveniles	Mean±SD	CV	% of control	Significance*
Control	1	170	152± 26.0	17.1	_	_
	2	141
	3	125
	4	123
	5	199
	6	167
	7	136
	8	156
0.5	1	182	153 ± 25.6	16.7	101	No
	2	167
	3	132
	4	131
1	1	128	145 ± 26.7	18.4	95.4	No
	2	154
	3	119
	4	178
2	1	10	6 ± 3.65	60.8	3.95	Yes
	2	8
	3	2
	4	4
4	1	0	0 ± 0.0	-	0.0	Yes
	2	0
	3	0
	4	0

Rep. = Replicate  a.s. = active substance  SD = Standard deviation      CV = Coefficient of variation

*) = Statistically significantly reduced compared tocontrol (Multiple Sequential-rejective Welsh-t-test After Bonferroni-Holm)

Validity criteria fulfilled:

yes

Conclusions:

During this study on the effects of p-xylene on earthworms, a NOEC of 71.1 mg/kg soil dry weight (based on initial measured concentrations, equivalent to a nominal 189 mg/kg soil dry weight) was determined based on mortality of adults and lack of juveniles produced at the top two doses tested (initial measured concentrations of 124 and 235 mg/kg soil dry weight, equivalent to nominal concentrations of 340 and 612 mg/kg soil dry weight, respectively). Following statistical analysis, an EC50 of 88.8 mg/kg soil dry weight (95 % confidence limits for range 81.3-97.1 mg/kg soil dry weight, based on the initial measured concentration) was determined based on significant decreases in the presence of juveniles and unhatched cocoons compared to controls.

Executive summary:

Effects of p-xylene on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222.

The study was conducted under static conditions over 8 weeks with p-xylene at initial measured concentrations of 0, 3.48, 7.04, 11.5, 22.2, 35.8, 71.1, 124 or 235 mg/kg soil dry weight (reported to be equivalent to nominal concentrations of 0, 10, 18, 32, 58, 105, 189, 340, and 612 mg/kg soil dry weight, respectively). Each application rate was mixed into the artificial soil containing 5 % peat one day prior to introducing earthworms to the soil. A control using untreated artificial soil was tested under the same conditions as the test item treatments. After approx. 24 hours of equilibration of the test item a total of 80 test organisms were introduced into 8 control replicates and 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.34 and 0.57 g at the beginning of the experiment. To reduce volatility of the test item the test containers were covered with opaque aluminium foil which was perforated on day 7 with 5 pin-sized holes per replicate to allow air exchange.

After 28 days of exposure in soil, neither earthworm mortalities nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or initial measured test item concentrations 3.48 to 71.1 mg/kg soil dry weight (reported to be equivalent to nominal concentrations of 10 to 189 mg/kg soil dry weight). However, at the top two doses 100 % mortality were observed. There were no statistically significant differences in earthworm body weights in the initial measured test item concentrations 3.48 to 71.1 mg/kg soil dry weight compared to the control group. Due to 100 % mortality at the top two doses, no body weights were measured. After a further four weeks, the mean reproduction rate (average number of juveniles produced per replicate) was 177 in the control and ranged from 0 to 193 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 3.48 to 71.1 mg/kg soil dry weight compared to the control. At the top two doses, earthworm reproduction was significantly reduced as no juveniles were found.

During this study on the effects of p-xylene on earthworms, a NOEC of 71.1 mg/kg soil dry weight (based on initial measured concentrations, equivalent to a nominal 189 mg/kg soil dry weight) was determined based on mortality of adults and lack of juveniles produced at the top two doses tested (initial measured concentrations of 124 and 235 mg/kg soil dry weight, equivalent to nominal concentrations of 340 and 612 mg/kg soil dry weight, respectively). Following statistical analysis, an EC50 of 88.8 mg/kg soil dry weight (95 % confidence limits for range 81.3-97.1 mg/kg soil dry weight, based on the initial measured concentration) was determined based on significant decreases in the presence of juveniles and unhatched cocoons compared to controls.

Description of key information

There are two earthworm reproduction studies available one for p-xylene and one for mixed xylenes (CAS 1330 -20 -7; substance composition equivalent to reaction mass of ethylbenzene and mixed xylenes). The study on p-xylene reports a NOEC of 71.1 mg/kg soil dry weight and an EC50 of 88.8 mg/kg soil dry weight (initial measured concentrations) based on mortality and effect on reproduction. The study on mixed xylenes (CAS 1330 -20 -7; substance composition equivalent to reaction mass of ethylbenzene and mixed xylenes) reported a NOEC of 42.6 mg/kg soil dry weight and an EC50 of 93.2 mg/kg soil dry weight (initial measured concentrations) based on effects on reproduction.

Due to the technical challenges of testing volatile substances, a TLM-EqP modeling framework was used to support terrestrial effect values derived experimentally and are presented as supporting studies. These data were considered reliable and comparable to experimentally derived values; however, were not used in the PNEC derivation in order to comply with ECHA FDL (2014) on using experimental data to derive PNEC. The hazard to soil organisms was assessed by using the experimental data.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

88.8 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

42.6 mg/kg soil dw

Additional information

Due to the technical challenges of testing volatile substances, a TLM-EqP modeling framework was used to support terrestrial effect values derived experimentally and are presented as supporting studies. The estimated 28 day LL50 values from TLM QSAR were 22.082 - 74.410 mg/kg dw and 56 day long-term effects NOELR values from TLM QSAR were 4.2 30 - 14.255 mg/kg dw.

The Draft Screening Assessment Report (Environment Canada 2014) reports a 14 day NOEC for earthworms of 16 mg/kg dw from ESG International (2002).